A. Daniel Jones, Ph.D. - US grants

The aim of the Analytical Chemistry Core is to support Program Project investigators with the development and application of sophisticated methods of chemical isolation, fractionation, structure determination, and quantitative analysis. The function of the Core is to provide cost- efficient means for individual laboratories to obtain access to expertise and capabilities not otherwise available in individual laboratories. The Core will subsidize the use of mass spectrometric, chromatographic, and spectroscopic techniques for the entire program project. Furthermore, the Core will support the development of improved analytical technologies including immunoaffinity isolation, multichannel near-IR fluorescence detectors, laser desorption MS, and atomic force microscopy (AFM). Specific efforts target development of near-IR fluorescence-based ELISAs for determination of biomarkers of exposure including mercapturic acid and other thioether metabolites. UV-visible spectroscopic and mass spectrometric techniques will be used to optimize hapten loadings on antigenic conjugates and enzyme tracers. GC, HPLC, GC/MS, and LC/MS techniques will be employed to confirm immunoassay results and validate assay performance. Synthetic intermediates will be characterized using high resolution MS, MS/MS, and NMR, Uv-vis, and FTIR spectroscopy. Support will be provided for studies of thermal and biological remediation by performing localization and quantitative speciation of organic, organometallic, and inorganic byproducts. Laser desorption mass spectrometry will be employed for rapid speciation of metals (e.g. chromium) and organic contaminants in particulate and solid samples including soils. Atomic absorption spectroscopy, stripping voltammetry, x-ray fluorescence, electron microscopy, and AFM will complement these efforts. Microcolumn HPLC/electrospray MS methods will be to characterize variants of human chorionic gonadotropin as candidate biomarkers of reproductive function, as well as metabolites of naphthalene, nitronaphthalene, and chlorinated ethanes and ethenes. Electrospray MS and laser desorption MS will be employed to characterize adducts of electrophilic metabolites with specific protein targets. Similar methods will be used to probe the structures and expression of key metabolic proteins such as esterases, hydrolases, and glutathione-S-transferase isozymes in target tissues and also in organisms used for bioassays and bioremediation. Bioassay-directed fractionation schemes including column chromatography and ultrafiltration will be developed to explore partitioning of organic and inorganic substances between vapor, particulate, colloidal, and dissolved phases as this partitioning is expected to have profound effects upon transport, bioavailability, and toxicity. The Analytical Core will also provide instruction and training to Superfund researchers in development of sampling protocols, experimental design, instrument operation, and data interpretation, and will act to facilitate collaborations between Superfund projects.

9512231 Chiang An advanced surface microscopy facility, consisting of an ultrahigh vacuum low energy electron microscope (LEEM) and a ultrahigh vacuum scanning tunneling microscope (STM) installed within the same vacuum system with sample transfer between the two instruments, will be established. The combination of the two instruments, LEEM and STM, within the same ultrahigh vacuum environment will enable the structural study of the surfaces of materials with both atomic resolution (STM) and in real time (LEEM). Both microscopes will permit control of the sample temperature during imaging and deposition of metals and adsorbates. The facility will be used by eleven investigators and their research groups to study surface properties in several key areas. These include: (1) dynamics of surface structural changes, such as nucleation and growth of metal, semiconductor, and oxide overlayers; (2) structural effects on adsorbates, such as segregation, diffusion, and reactivity; (3) synthesis and characterization of nanoscale materials, such as quantum dots, thin-film fullerene polymers, and nanoclusters. %%% Two complementary types of surface microscopes, a low energy electron microscope and a scanning tunneling microscope, will be utilized to probe surface systems with varying spatial and time resolution as a function of important parameters such as temperature and coverage of adsorbates. The possibility exists to image an important class of catalysts and simultaneously investigate their reactivities and catalytic properties. ***

The Analytical Core is a central resource designed to facilitate the development and application of powerful analytical methods to solve key problems encountered by the components of the Superfund Project. The general goals of the Analytical Core are to facilitate research and develop analytical technologies that address the detection, assessment, evaluation and reduction of hazardous chemical exposure and the associated population health risks. There are three specific aims proposed by the Analytical Core: 1) Provide analytical support for projects in the Superfund Program using chromatographic and spectroscopic techniques. 2) Provide high sensitivity analysis of hazardous substances using Accelerator Mass Spectroscopy (AMS) and Nuclear Microscopy. 3) Provide support in peptide chemistry including gas phase sequencing and LC-MS analysis of peptides. Accomplishment of Aim 1 will include enhanced development of phase sequencing of chemical exposure through application of immunoaffinity purification and development of HPLC/MS/MS biomarkers of chemical exposure through application of immunoaffinity purification and development of HPLC/MS/MS driven multi-dimensional metabolite profiling. Support will also be provided by for toxic substances identification from complex mixtures by performing fractionation and structural elucidation duties in multi-project toxicity identification complex mixtures by performing fractionation and structural elucidation duties in multi-project toxicity identification evaluation studies (TIEs). The Analytical Core will develop vacuum ultraviolet ionization mass spectroscopy instrumentation for the sensitive detection and quantitation of toxic metal oxides and polyhalogenated aromatic hydrocarbons produced during incineration and linked to human health risks. Aim 2 will utilize the attomole (i.e. 10-18) sensitivity of AMS to improve the analyses of risks to human health due to hazardous chemicals in the environment. Chemicals labeled with low levels of 14C, 36Cl, and other long-lived isotopes will be traced quantitatively through ecosystems, animal models, and humans. Using HPLC separation the kinetics, the transformations and final biological targets of these chemicals including proteins and DNA will be identified. AMS will also be used to enhance the sensitivity of developed immunochemical biomarker detection. Nuclear Microprobe analyses will also be used to quantify the location, distribution, and amount of heavy metals from the environment of filters or tissue that can similarly lead to diseased states. Aim 3 will address the questions of protein identify and modification associated with hazardous chemical exposure. HPLC/MS/MS, enzymatic digestion and peptide mapping will be the primary tools for the accomplishment of this aim. The Core scientists will educate Superfund research scientists in the use And value of the full array of analytical techniques.

Baculoviruses are a group of viruses that infect insects. The effect of these viruses on insects, however, appears to vary with the chemical composition of the host plant that the insect consumes. For example, baculoviruses are less able to kill tobacco budworm (Heliothis virescens) when the insect feeds on cotton, than when it feeds on its other host plants such as tomato or lettuce.

In this study, the PIs seek to determine the chemical interactions that cause some host plants to render their insect herbivores more resistant to infection by baculoviruses. Preliminary studies have suggested that the generation of reactive oxygen species (ROS) is a major cause of such disease inhibition, but it is unclear how this occurs or what steps in the pathway of infection are being altered by these reactive products. Generation of ROS occurs as a consequence of normal cellular processes but can be altered by ingestion of dietary chemicals. High levels of ROS lead to a condition known as oxidative stress which can damage cellular substances and has been associated with aging and cancer development. ROS can also destroy many microorganisms and appear to interfere with the ability of baculoviruses to kill their host insects. This study will examine whether diet-related ROS interfere with viral infection of insects by damaging the virus or by altering insect immune responses.

These investigations should have broad implications for understanding the chemical basis of complex interactions among multiple trophic levels. Additionally, results will contribute to a general understanding of the effects of oxidative stress on disease processes and disease ecology. Finally, because baculoviruses are currently being used as an environmentally-friendly alternative to chemical pesticides, these results may help improve biological control practices in agriculture.

Studies of the molecular bases of plant responses to enemies have focused more on microbes than on insects, despite the great ecological, evolutionary, and economic impacts of herbivores. Research on plant responses to insects has been limited mainly to phenotypic (chemical) defenses. Little attempt has been made to link molecular and chemical responses to either type of pest to understand the mechanisms underlying plant defense.

This project will employ microarrays of approximately 2500 stress-responsive genes (developed by a previous NSF Plant Genome project) plus critical genes in the glucosinolate biosynthetic pathway. Gene expression and glucosinolate profiles will be developed for plants responding to insect species and pathogens representing chewing and sucking feeding types and a range of specialization on Brassicaceae. The major goal of this limited proof-of-concept project is to demonstrate that gene function can be inferred by using multivariate statistical treatments to link array results to defense metabolite response profiles and to ecological outcomes. "Gene function" in terms of defense will be inferred in terms of expression pattern in relation to known functions (e.g., in signaling pathways, metabolite synthesis) and in the context of the ecological outcome (e.g., impact on insect, resistance to herbivory) using standard multivariate clustering techniques, concordance analysis, and Mantel covariate statistics. Identities and functions of critical components of plant defense responses identified statistically will eventually be confirmed using mutants and knockouts in bioassays with specific insects and microbes under varying environmental conditions. Images of expression patterns, metabolite profile data, integrative results, protocol updates, and instructions for obtaining material will be posted monthly at the following TAIR-linked URL: http://schultzlab.cas.psu.edu/ . Metabolomics data will be archived at the Genomic Arabidopsis Resource Network (http://www.york.ac.uk/res/garnet/beale.htm) and functional genomics data at TAIR (http://www.arabidopsis.org/info/2010_projects/ ).

Arabidopsis 2010 objectives addressed by this project include determining the functions of genes in the context of plant defense, producing a stress microarray and chemical analytical protocols as tools useful to a wide range of investigators, and developing statistical protocols molecular ecologists can use to link genotypic and phenotypic responses. In addition, the project will be coordinated with 2 existing Arabidopsis 2010 programs (Wurtele et al. "Visual informatics tools" and Lewis et al. "Phenylpropanoid metabolite networks") and establishes an international collaboration with the Max Planck Institute for Chemical Ecology (Jena, Germany).

Broader impacts of the limited project will include training 3 graduate students, exploiting existing K-12, teacher, and undergraduate research experience links that already exist in the PSU College of Agricultural Sciences and Chemistry Department, and developing platforms for using Arabidopsis responses as a teaching tool in the K-8 environment.

PI: Robert L. Last (Michigan State University)

CoPIs: David R. Gang (University of Arizona), Gregg A. Howe (Michigan State University), A. Daniel Jones (Michigan State University), Eran Pichersky (University of Michigan), and Curtis Wilkerson (Michigan State University)

Collaborators: HyeRan Kim (University of Arizona), Kenneth Nadler (Michigan State University), and Carol A. Soderlund (University of Arizona)

The long term goal of this project is to lay a foundation for a complete 'systems biology' understanding of the entire network of genes and proteins involved in the development of each of the different types of glandular trichomes found in tomato and related species in the Solanum and the full set of genes and enzymes responsible for their biosynthetic capacity. The glandular trichomes are chosen as the focus of this project because these fascinating cell-surface structures make a wide variety of structurally and biosynthetically diverse small molecules. These specialized (secondary) products come from many different biosynthetic pathways and are known or postulated to serve a variety of roles in stress adaptation, including providing defense against important plant pests. Comparison of genes found from the EST sequences and metabolites found in the different gland types will be used to discover new biosynthetic enzymes, and these will be tested for function by in vitro biochemical methods and by in vivo methods using transgenic plants. Mutants and introgression lines will be evaluated for changes in glandular trichome morphology and chemistry and genetic analysis initiated for future identification of genes that are responsible for these novel phenotypes.

Broader Impacts

These studies should inform breeding and transgenic approaches to improving stress tolerance in agriculturally important plants. The data from this project will be provided to GenBank, The SOL Genomics Network (http://sgn.cornell.edu/) and made available through a project website. This project will integrate research and education in three ways: 1. Summer research experiences for undergraduate students and secondary school teachers; 2. Training of students in successful approaches to doing research in an interdisciplinary and geographically dispersed environment, which is becoming more and more important for success in the biological sciences; 3. Training of participants in cutting-edge, computer-based curriculum development tools with the Lon-CAPA web-based course management system developed at Michigan State University. A strong emphasis will be placed on recruitment and training of underrepresented minorities and women.

This award is for the acquisition of a mass spectrometer (a triple quadrupole LC/MS/MS system) and a nitrogen generator for trace level structure-based screening and quantitative profiling of nonvolatile metabolites and reaction products. This system will enable sensitive and high-throughput non-target (metabolomics) and target metabolite analyses. Biology is moving toward a new and interdisciplinary paradigm of systems biology, which seeks to replace the one gene, pathway, or physiological process-at-a-time approaches to understanding complex biological systems with a more efficient and holistic concept. This approach requires the production and datamining of comprehensive and high quality data sets describing dynamic changes in genome, transcriptome, proteome and metabolome. The instruments acquired through this award will allow the investigators to take advantage of the great technological advances that have been made in methods for analysis of metabolites, including the global approach termed metabolomics, largely due to improvements in Mass Spectrometry in recent years.

This mass spectrometer was chosen because it is user friendly, for use by undergraduate and graduate students, as well as postdoctorals and faculty. Individuals trained in mass spectrometer operation, analytical method design, and data interpretation will represent a good gender balance and come from a variety of ethnic, cultural, and educational backgrounds. This is due to recruitment infrastructure on campus and a group of participating faculty who are culturally and ethnically diverse and who seek to train a diverse group of students. Training sessions and workshops will introduce students to the problem-solving power of mass spectrometry, which is one of the most dynamic fields of analytical chemistry. Use of these instruments will accelerate research and improve understanding of gene functions, responses of plants, animals, and microbes to changing environments, and the chemistry governing interactions between organisms.

PI: Robert Last (Michigan State University)

CoPIs: Cornelius Barry and A. Daniel Jones (Michigan State University) and Eran Pichersky (University of Michigan)

The long-term goal of this research is to understand the principles that underlie the tremendous diversity of plant specialized metabolites (also known as secondary metabolites or natural products). These chemicals are part of the plant's arsenal for defense against biological and environmental stress and include compounds that contribute to flavors and aromas as well as medically important drugs. Storage/secreting glandular trichomes (SGTs) on the aerial surfaces of the plant are especially active in producing such compounds and thus are excellent for studying their biosynthesis. Cultivated tomato (Solanum lycopersicum) and related species have anatomically diverse types of SGTs on leaves, stems, and reproductive structures and each type produces a distinct set of specialized metabolites including terpenes, methylated or glycosylated flavonoids, and acylsugars, with surprising diversity observed within and between species. It is becoming increasingly clear that Solanum SGTs utilize novel biochemical mechanisms for producing some of these products, and recent results indicate that the combination of comparative and functional genomics, metabolomics, and biochemical methods is a particularly promising approach to elucidate these pathways and the overall pattern of their diversification. Building on the progress made in the previous funding period, this project will provide a detailed analysis of the biosynthesis of terpenes and acylsugars, two classes of compounds with documented biological effects against biotic stress agents, in distinct types of SGTs. The approach is designed to elucidate specific reactions and enzymes but also patterns of pathway evolution and regulation. Acylsugar biosynthesis is currently poorly understood, and the genetics and genomics resources created under this project will provide valuable insight into how these diverse compounds are synthesized. More is known about terpene biosynthesis, but previous results indicated that some of the tenets about substrate utilization and roles of specific classes of terpene synthases are imperfect and instead these proteins display a tremendous potential to evolve new functions, a process that will be examined in this project.

The broader impacts of this project fall into two general categories: advancement of the fields of biology impacted by the project and educational outreach. The results of these studies will reveal the biochemical and genetic mechanisms by which plants produce these important compounds in SGTs and other cell types, and inform breeding and transgenic approaches to modify their synthesis in crop plants to enhance resistance to insects and disease. Discovery of novel mechanisms also offers opportunities to exploit these biochemical pathways in new technologies for the production of bioactive chemicals and chemical feedstocks. In order to maximize the utility and accessibility of the data generated through this project, data will be submitted to long-term community databases that include the NCBI (http://www.ncbi.nlm.nih.gov/), Trichome (http://www.trichome.msu.edu/), TrichOME (http://www.planttrichome.org/trichomedb/) and the Sol Genomics Network (http://solgenomics.net/). Because the project includes laboratories expert in analytical chemistry, biochemistry, genetics and genomics, it provides a natural platform for interdisciplinary training. In addition to cross-training of undergraduate and graduate students and postdoctoral researchers throughout the year, this project will integrate research and education in complementary ways: first, through summer research experiences for three undergraduate students each year as part of the Plant Genomics @ Michigan State University program which places an emphasis on the recruitment of women and underrepresented minority group members; and second, through training opportunities for faculty from primarily undergraduate schools.

Part 1: Non-technical abstract

Plants are master chemists, producing thousands of small molecules of varied structures and activities. Some of these specialized metabolites have well established roles, including protection from diseases and insects and attraction of beneficial partner organisms. Some are used by humans as medicines and environmentally safe pesticides. The metabolic pathways for only a small fraction of these compounds are well understood, leaving much to learn about how plants produce this enormous diversity of products. This research will focus on specialized metabolism in the Solanaceae (nightshade) family, which includes the important crops tomato, potato, peppers and eggplant and in which a great diversity of natural products is documented. The overarching goal is to develop computational and experimental approaches to discover new plant chemicals and to find the genes that plants use to make small molecules that are valuable for agriculture and human wellbeing. The project outcomes will expand the understanding of the biochemical and genetic mechanisms by which plants produce different classes of specialized metabolites. This research will support breeding and transgenic approaches to improve specialized metabolite synthesis in crop plants to increase resistance to disease and insects and enhance crop value; it will also develop new methods for combining computational and experimental approaches in the study of metabolism. The project outreach activities include summer research for undergraduates from under-represented groups, training of faculty for primarily undergraduate institutions with substantial minority enrollments, and a summer program for science outreach to adults.


Part 2: Technical abstract

The identification of genes involved in specialized metabolism is of great importance, since changes in these genes provide a basis for lineage-specific chemical diversity. This project will provide quantitative assessments of the differences between specialized metabolism genes and other genes. The predicted portion of the genome devoted to specialized metabolism within the Solanaceae will be tested using hypothesis-driven experimental approaches. This analysis of the Solanaceae family, which includes important crops as well as models in plant ecology and evolution, will establish a paradigm for computationally predicting and experimentally validating specialized metabolism-related genes across the plant kingdom. The project will take advantage of the rapidly increasing plant genome and transcriptome resources in the Solanaceae to define computationally the characteristics of genes encoding specialized metabolic enzymes. The computational approaches will be coupled with analytical chemical methods, including mass spectrometry and nuclear magnetic resonance spectroscopy, to discover specialized metabolites and to guide the identification of candidate genes encoding enzymes that produce novel metabolites. In vitro protein biochemistry and functional genomics methods will be employed to validate gene candidate functions, and to improve the accuracy of the computational methods. The project outreach activities include summer research for undergraduates from under-represented groups, training of faculty for primarily undergraduate institutions with substantial minority enrollments, and a summer program for science outreach to adults.